Effect of miR-203 expression on myocardial fibrosis.

OBJECTIVE: Cardiovascular disease is one of the diseases threatening human health. Myocardial fibrosis is a major cause of cardiovascular diseases. Studies have shown that over expression of miR-203 can inhibit the fibrosis. Therefore, in this study, the effect of differential expression of miR-203 on fibrosis of cultured mouse cardiomyocytes was investigated. MATERIALS AND METHODS: Activators and inhibitors of miR-203 were designed according to the sequence of miR-203, synthesized, and transfected into mouse cardiomyocytes to establish activator group, inhibitor group, and control group. The expression levels of fibrosis-related factors including FN, CTGF, and TGF-ß1 were measured by Western blot and RT-PCR 24 h and 36 h after transfection. RESULTS: Over-expression of miR-203 in mouse cardiomyocytes significantly decreased the expression levels of TGF-ß1, CTGF, and FN in a time-dependent manner, compared with that in the control group (p <0.05). Inhibition of miR-203 expression in mouse cardiomyocytes significantly increased the expression levels of TGF-ß1, CTGF, and FN 36 h after transfection, compared with that in the control group (p < 0.05). No significant differences were seen in the expression levels of TGF-ß1, CTGF, and FN 24 h after transfection, compared with that in the control group (p >0.05). CONCLUSIONS: Over-expression of miR-203 in mouse cardiomyocytes significantly decreased the expression levels of TGF-ß1, CTGF, and FN, which might be used as a detection index for prediction of fibrosis.

Incorporation of quantum dots in silk biomaterials for fluorescence imaging.

Tracking the distribution and degradation of biomaterials after in vivo implantation or injection is important for tissue engineering and drug delivery. Intrinsic and externally labeled fluorescence has been widely used for these purposes. In the present study, 3-mercaptopropionic acid (MPA)-coated CdTe quantum dots (QDs) were incorporated into silk materials via strong interactions between QDs and silk, likely involving the hydrophobic beta-sheet structures in silk. MPA-QDs were pre-mixed with silk solution, followed by ultrasonication to induce silk gelation or by blending with polyvinyl alcohol (PVA) to generate silk microspheres. Silk structural changes and hydrogel/microsphere morphologies were examined by ATR-FTIR and SEM, respectively. The fluorescence of QDs-incorporated silk hydrogels and microspheres remained stable in PBS pH 7.4 for more than 4 days. The amount of QDs released from the materials during the incubation was dependent on loading; no QDs were released when loading was below 0.026 nmol/mg silk. After subcutaneous injection in mice, the fluorescence of QDs-incorporated silk microspheres was quenched within 24 h, similar to that of free QDs. In contrast, the QDs-incorporated silk hydrogels fluoresced for more than 4 days in vivo.

Efficacy of chlortetracycline treatment on vulvar non-neoplastic epithelial disorders.

OBJECTIVE: To observe the effectiveness of chlortetracycline (aureomycin) treatment on vulval white lesions and to explore its possible pathogenesis. MATERIALS AND METHODS: From January 2001 to April 2011, 194 patients with vulvar non-neoplastic epithelial disorders were divided into three groups according to therapy regimens received, ie, chlortetracycline treatment group (72 cases), chlortetracycline + beclomethasone treatment group (66 cases), and beclomethasone treatment group (56 cases); their local changes of vulvar lesions were observed and efficacy of these treatment profiles was evaluated after one year. RESULTS: Effective rates of chlortetracycline group, chlortetracycline + clobetasol group and clobetasol groups were 86.1% (62/72), 87.9% (58/66), and 62.5% (35/56), respectively. There was a significant difference among these three groups (Hc = 10.7766,p = 0.0046), the curative rate of clobetasol group was markedly lower than that of the former two groups (p = 0.0072 and p = 0.0019), but was not statistical significant (p = 0.6077) when compared between the former groups. CONCLUSION: The occurrence of vulvar non-neoplastic epithelial disorders may be associated with chlamydia and mycoplasma infection, the chlortetracycline is an effective drug for this illness, the mechanism of which might be related to killing pathogens directly and inhibiting inflammatory mediators.

Alteration of T-cell subpopulations and lipid peroxidation in the blood of patients with vulvar non-neoplastic epithelial disorder.

OBJECTIVE: To determine the relationship between vulvar non-neoplastic epithelial disorder and thymus-dependent lymphyocyte levels and lipid peroxidation. MATERIALS AND METHODS: the authors measured the levels of CD3+, CD4+, CD8+, CD16+ T cell, and the concentration of superoxide dismutase (SOD) and malondialdehyde (MDA) in the blood of 62 patients with vulvar non-neoplastic epithelial disorder. A control group consisted of 30 normal women from the present hospitals. RESULTS: The level of CD4+/CD8+ T-lymphocytes and SOD in the blood of the patients with vulvar non-neoplastic epithelial disorder was significantly lower than that in control subjects, but the level of MDA was higher as compared with normal women. CONCLUSION: There is increased immune activation and lipid peroxidation in patients with vulvar non-neoplastic epithelial disorder, which could contribute to destruction of vulvar tissue.

Exogenous growth hormone attenuates cognitive deficits induced by intermittent hypoxia in rats.

Sleep disordered breathing (SDB), which is characterized by intermittent hypoxia (IH) during sleep, causes substantial cardiovascular and neurocognitive complications and has become a growing public health problem. SDB is associated with suppression of growth hormone (GH) secretion, the latter being integrally involved in the growth, development, and function of the CNS. Since GH treatment is able to attenuate neurocognitive deficits in a hypoxic-ischemic stroke model, GH, GH receptor (GHR) mRNA expression, and GH protein expression were assessed in rat hippocampus after exposures to chronic sustained hypoxia (CH, 10% O(2)) or IH (10% O(2) alternating with 21% O(2) every 90 s). In addition, the effect of GH treatment (50 µg/kg daily s.c. injection) on erythropoietin (EPO), vascular endothelial growth factor (VEGF), heme oxygenase-1 (HO-1), and GLUT-1 mRNA expression and neurobehavioral function was assessed. CH significantly increased GH mRNA and protein expression, as well as insulin-like growth factor-1 (IGF-1). In contrast, IH only induced a moderate increase in GH mRNA and a slight elevation in GH protein at day 1, but no increases in IGF-1. CH, but not IH, up-regulated GHR mRNA in the hippocampus. IH induced marked neurocognitive deficits compared with CH or room air (RA). Furthermore, exogenous GH administration increased hippocampal mRNA expression of IGF-1, EPO, and VEGF, and not only reduced IH-induced hippocampal injury, but also attenuated IH-induced cognitive deficits. Thus, exogenous GH may provide a viable therapeutic intervention to protect IH-vulnerable brain regions from SDB-associated neuronal loss and associated neurocognitive dysfunction.

Use of genetically modified allograft to deliver local immunomodulatory molecule with minimal systemic toxicity in a rat model of allogeneic skin flap transplantation.

The effects of OX40-OX40 ligand (OX40L) costimulatory pathway blockade to prevent T-cell-mediated acute rejection were investigated in a rat model of allogeneic superficial inferior epigastric artery flap transplantation. An ex vivo gene transfer technique was used to modify allografts to locally deliver an immunomodulatory molecule. The flaps were separated from donors, perfused with an adenoviral vector expressing the OX40 immunoglobulin (AdOX40Ig) for 1 hour, and incubated at 37°C for 2 hours. Before transplantation, the flaps were flushed with phosphate-buffered saline solution to remove unincorporated viral particles. Recipients were randomly divided into 5 groups, and treated with topical OX40Ig gene transfer, a single low dose of rapamycin alone, or a combination of agents. Graft survival was assessed using histopathologic classification of skin rejection. All animals in the untreated group (n = 9) or the group treated with adenovirus expressing green fluorescence protein (n = 9) developed grade 3 clinical rejection by postoperative day 7. No significant difference was observed in graft survival between the locally treated AdOX40Ig groups (mean [SD], 8.1 [0.7] days) and the untreated groups (7.7 [1.2] days) could be observed (P > .05, t test). Graft survival in the locally treated AdOX40Ig groups was extended to 18.7 (1.2) days when transduction was combined with a low dose of rapamycin, a significant improvement over survival with rapamycin treatment alone (13.2 [0.6] days) (P < .01). These results demonstrated that local immunomodulation by the allograft itself and low-dose rapamycin treatment promote graft acceptance. This protocol may enable reduction of the dosage of immunosuppressive drugs needed for successful inhibition of acute rejection in the early postoperative period.

Preparation of the acellular scaffold of the spinal cord and the study of biocompatibility.

STUDY DESIGN: Acellular spinal cord was prepared through chemical extraction, and its biocompatibility was studied. OBJECTIVE: Acellular scaffolds have been developed from various materials for tissue reconstruction, except for spinal cord. The objective of this study was to prepare acellular spinal cord and examine the biocompatibility of the scaffold. SETTING: This study was conducted at the Department of Orthopedics, Xinqiao Hospital, The Third Military Medical University, Chongqing, China. METHODS: The morphology of the acellular segments was revealed by scanning electron microscopy, immunohistochemistry, and hematoxylin and eosin stain. Biocompatibility was studied by immunohistochemistry. RESULTS: Results show that in spinal cord scaffolds, cells, myelin sheath and axon of nerve fibers were eliminated, and three-dimensional supports of extracellular matrix were reserved. The component analytical results of the acellular spinal cord indicate that they contain laminin, fibronectin and collagen, which can facilitate and induce the regeneration of injured nerves, and enhance the adhesion and proliferation of cells. The acellular spinal cord has a three-dimensional structure and excellent biocompatibility. CONCLUSION: Our data indicate that acellular spinal cord has certain biological properties and it may be a potential alternative scaffold for spinal cord tissue engineering.

A very rare complication: new hair growth around healing wounds.

We present the case of a patient in whom active new hair growth occurred around a wound after healing. This very rare phenomenon has not previously been reported in the literature. We postulate that, after the epidermis and hair follicles have been damaged by wounding, it is possible for them naturally to heal and repair if provided with an appropriate chemical and physical microenvironment. This hypothesis may inspire new thinking in the management of alopecia, tissue engineering and the regeneration of other organs.

Troglitazone suppresses transforming growth factor-beta1-induced collagen type I expression in keloid fibroblasts.

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR)-gamma agonists are increasingly used in patients with diabetes and some studies have suggested a beneficial effect on organ fibrosis. However their effects on dermal fibrosis in keloids are unknown. OBJECTIVE: To investigate the effect of the PPAR-gamma agonist troglitazone on transforming growth factor (TGF)-beta1-induced collagen type I expression in keloid fibroblasts. METHODS: Keloid fibroblasts were cultured and exposed to different concentrations of troglitazone in the presence of TGF-beta1. The mRNA expression of PPAR-gamma was determined by semiquantitative reverse transcriptase-polymerase chain reaction. The protein of PPAR-gamma, Smad2, Smad3, phoshpo-Smad2/3 and collagen type I was determined by Western blotting and collagen synthesis was evaluated by measuring (3)H-proline incorporation. The effect of troglitazone on cell viability was evaluated by the colorimetric conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. RESULTS: PPAR-gamma was expressed at a moderate level in keloid fibroblasts. Troglitazone depressed TGF-beta1-stimulated collagen type I expression and collagen synthesis in keloid fibroblasts in a concentration-dependent manner. Moreover, troglitazone inhibited expression and phosphorylation of TGF-beta1-induced Smad2/3. Cell viability was unaffected. These inhibitory effects of troglitazone were reversed by the PPAR-gamma-specific antagonist GW9662. CONCLUSIONS: Our data suggest that PPAR-gamma is present in keloid fibroblasts and PPAR-gamma activation inhibits TGF-beta1-induced collagen type I expression at least in part by decreasing collagen synthesis. PPAR-gamma may be a promising therapeutic target for keloids.

VEGF gene therapy for the survival of transplanted fat tissue in nude mice.

The objective of this study was to determine the effects of adenovirus-mediated vascular endothelial growth factor (Ad-VEGF) on the angiogenesis and survival of free-fat tissue transplantation in nude mice. Thirty 6-week-old CD-1 nude male mice were injected with 1ml fat tissue (harvested by suction-assisted lipectomy from the breast of humans) in the subcutaneous of scalp and were randomised into three groups of 10 animals each. Group 1 was the study group, in which Ad-VEGF was mixed with transplanted fat tissue and injected into mice. In group 2, adenovirus-mediated green fluorescent protein (Ad-GFP) gene was mixed with transplanted fat tissue and injected into the mice. In group 3, normal saline alone was used. Both group 2 and group 3 are control groups. The animals were euthanised 15 weeks after the procedure. The fat survival weight and volume of the study group were significantly greater than those of two control groups (p<0.05). Light microscopical examination of haematoxylin and eosin-stained slides of the dissected fat 15 weeks after injection was performed in group 1 and group 2. Less cyst formation and fibrosis, indicating improved quality of the injected fat, can be obtained by the addition of Ad-VEGF. Vascular density was evaluated at the microvascular level through the use of light microscopic sections of the central part of the fat tissue at 15 weeks after injection by von Willebrand factor staining. Histological evaluation showed that capillary density increased markedly in the study group mice. Mice of the study group disclosed significantly higher VEGF protein levels detected by ELISA assay of plasma samples obtained from the mice after the fat injection (day 1, 4, 7 and 28; p<0.01) at each time point than the mice of the two control groups. The findings reported in this study indicate that the VEGF gene therapy can enhance the survival and the quality of grafted fat tissue, which may be due to induction of angiogenesis.

Intermittent hypoxic exposure during light phase induces changes in cAMP response element binding protein activity in the rat CA1 hippocampal region: water maze performance correlates.

Intermittent hypoxia (IH) during sleep, a characteristic feature of sleep-disordered breathing (SDB) is associated with time-dependent apoptosis and spatial learning deficits in the adult rat. The mechanisms underlying such neurocognitive deficits remain unclear. Activation of the cAMP-response element binding protein (CREB) transcription factor mediates critical components of neuronal survival and memory consolidation in mammals. CREB phosphorylation and DNA binding, as well as the presence of apoptosis in the CA1 region of the hippocampus were examined in Sprague-Dawley male rats exposed to IH. Spatial reference task learning was assessed with the Morris water maze. IH induced significant decreases in Ser-133 phosphorylated CREB (pCREB) without changes in total CREB, starting as early as 1 h IH, peaking at 6 h-3 days, and returning toward normoxic levels by 14-30 days. Double-labeling immunohistochemistry for pCREB and Neu-N (a neuronal marker) confirmed these findings. The expression of cleaved caspase 3 (cC3) in the CA1, a marker of apoptosis, peaked at 3 days and returned to normoxic values at 14 days. Initial IH-induced impairments in spatial learning were followed by partial functional recovery starting at 14 days of IH exposure. We postulate that IH elicits time-dependent changes in CREB phosphorylation and nuclear binding that may account for decreased neuronal survival and spatial learning deficits in the adult rat. We suggest that CREB changes play an important role in the neurocognitive morbidity of SDB patients.

Experience with ketamine and sodium pentobarbital as anesthetics in a rat model of cardiac arrest and resuscitation.

We review 7 years experience with the chest compression model of cardiac arrest and resuscitation, comparing two different anesthetics. Ketamine stimulates cardiac function and only mildly depresses respiration; of the two it provides easier resuscitation. However, ketamine severely depresses brain protein synthesis; in studies using this measure ketamine is unsuitable and another agent must be used. Sodium pentobarbital mildly depresses brain protein synthesis, but depresses both cardiac and respiratory function, making resuscitation more difficult. Use of alternate chest/abdominal pumping (Babbs resuscitation technique), with judicious use of intra-cardiac epinephrine (adrenaline), made resuscitation reliable under sodium pentobarbital as well.

The initiating factors of late preconditioning in skeletal muscle.

PURPOSE: The goal of these studies was to determine the initiating factors for late preconditioning in the microcirculation of skeletal muscle. MATERIALS AND METHODS: The cremaster muscle of male Sprague-Dawley rats underwent 4 h of ischemia and then 60 min of reperfusion. Ischemic preconditioning (IPC) consisted of 45 min of ischemia but was done 24 h before the 4 h of ischemia. To mimic the effects of IPC in the late phase, adenosine (ADO) or sodium nitroprusside (SNP) was given 24 h before the prolonged ischemia via local intraarterial infusion. To block the effects of IPC in the late phase, 8-sulfophenyl-theophylline (a nonspecific ADO receptor blocker) or N(W)-nitro-l-arginine (a nonselective nitric oxide synthase antagonist) was given prior to IPC. Microvascular response to IPC and pharmacological preconditioning were determined by measuring arteriole diameters and capillary perfusion using intravital microscopy. RESULTS: Administration of ADO or SNP on day 1 without IPC produced a similar microvascular protection against prolonged ischemia/reperfusion on day 2 as that induced by IPC alone. In contrast, blocking ADO receptors or nitric oxide synthase on day 1 just prior to IPC eliminated the IPC-induced microvascular protection seen on day 2. In addition, inhibition of nitric oxide synthase on day 1 diminished the protection induced by ADO, but blocking ADO receptors on day 1 did not compromise the protection induced by SNP. CONCLUSION: The results from these studies suggest that up regulation of ADO is the initiating factor with secondary up regulation of nitric oxide in late preconditioning. Both ADO and nitric oxide contribute to initiating microvascular protection in the late phase of IPC.

[Experimental study on closing the firearm injured soft tissue defect by skin stretch].

OBJECTIVE: To investigate the availability and effect of skin stretch in closing the firearm injured soft tissue defect. METHODS: Eight white pigs with firearm injured soft tissue defect were divided into 3 groups. Each group I and group II had 3 pigs which were performed skin stretch. The control group had 2 pigs without stretch. The average diameter of the defect in three groups was (7.3 +/- 0.2) cm, (9.1 +/- 0.3) cm, (7.3 +/- 0.2) cm respectively, and the site of defect was on the lateral thigh and buttock. RESULTS: Skin stretch could make a visible reduction of the wound. It was possible to close the wound by direct traction when the diameter of the buttock wound was less than 7 cm, and when the diameter of the lateral thigh wound was less than the radius of thigh. The skin stretch should not last more than 7 days and the best effect appeared in 4 to 5 days after performing the skin stretch. CONCLUSION: The skin stretch can be applied in the repair of the firearm injured soft tissue defect. It has many advantage compared with the tradtional treatment.

[Effect of subcutaneous tissue trimming on the survival skin area of avulsion skin flap].

OBJECTIVE: To investigate the effect of subcutaneous tissue trimming on the survival skin area of avulsion skin flap. METHODS: Degloving injury was created in bilateral hind limbs of 7 pigs with avulsion injury machine, 4 cm x 10 cm avulsion skin flaps were elevated in degloving areas. Skin flaps in one side were replanted as control without any treatment. Subcutaneous tissue in the skin flaps of another side was partially excised and replanted by trimmed skin flaps. Survival skin flaps was calculated with computer at 7 days after operation. RESULTS: In the control group, the survival skin area was (40.41 +/- 9.23)%, while in the experimental group, the survival skin area was (60.90 +/- 15.26)%. There was significant difference between the two groups (P < 0.05). CONCLUSION: Trimming off subcutaneous tissue does improve the survival area of avulsion skin flap.

Initiation of microvascular protection by nitric oxide in late preconditioning.

The authors hypothesized that nitric oxide is induced by a brief period of ischemia/reperfusion (ischemic preconditioning, IPC) on postoperative day (POD) 1, and that this released nitric oxide is responsible for initiating a delayed microvascular protection against a prolonged period of ischemia in skeletal muscle on POD day 2. The cremaster muscle of male Sprague-Dawley rats underwent 4 hr of ischemia, and then 60 min of reperfusion. IPC consisted of 45 min of ischemia but was done 24 hr before the prolonged ischemia. Local intraarterial infusion of sodium nitroprusside (SNP, a donor of nitric oxide) or Nw-nitro-L-arginine (L-NA, a nonselective nitric oxide synthase antagonist) were also given 24 hr before prolonged ischemia. Arteriole diameters and capillary perfusion were measured using intravital microscopy. Four groups were compared: 1) control; 2) IPC; 3) SNP + sham IPC; and 4) L-NA + IPC. Four hours of ischemia followed by reperfusion created a significant vasoconstriction and capillary no-reflow in the microcirculation of cremaster muscles. These alterations were largely prevented by IPC. Local intraarterial infusion of SNP without IPC created a similar microvascular protection to that induced by IPC alone. In contrast, intraarterial infusion of L-NA prior to IPC eliminated the IPC-induced microvascular protection. In conclusion, in late preconditioning, nitric oxide contributes to the initiation of a delayed microvascular protection against prolonged ischemia in skeletal muscle.

Agents which block potassium-chloride cotransport prevent sound-triggered seizures in post-ischemic audiogenic seizure-prone rats.

The purpose of this study was to determine whether the loop diuretics furosemide, bumetanide and ethacrynic acid, which block the KCC1 potassium-chloride transporter in the kidney loop of Henle and the KCC2 potassium-chloride transporter in neuronal membranes, would prevent sound-triggered seizures in post-ischemic audiogenic seizure-prone rats. The rats were infused with the test agent via tail vein shortly before being tested for seizure susceptibility by exposure to loud noise (an alarm bell) for 60 s. Sound exposures were repeated at intervals to determine the time course of the seizure suppression effect. All three loop diuretics suppressed sound-triggered seizures in post-ischemic rats tested 2 days to 4 weeks after the ischemic exposure. Furosemide 200 mg/kg had no effect in 4/4 rats made acutely audiogenic seizure-prone by infusion of bicuculline into the inferior colliculus, indicating that the effect was not due to general anti-seizure activity. Mannitol 2 g/kg had no effect in 6/6 post-ischemic rats, indicating that the effect was not due to diuresis or fluid shifts. These results are consistent with the hypothesis that the exposure to global ischemia caused an upregulation of the potassium-chloride transporter KCC2 in neurons which persisted for at least 4 weeks.

A case study of ligation induced calcification in middle cerebral artery in rat.

A 90 min ligation of the middle cerebral artery (MCA) followed by 72-hour reperfusion appeared to cause calcium deposition in vascular myocytes of the tunica media and the perivascular tissue of the Sprague Dawley rat. The presence of small ovoid to large irregularly shaped intracellular opaque deposits were demonstrated by light and electron microscopy. Using X-ray elemental analysis the chemical nature of the deposits was found to be calcium phosphate. The functional significance of this first demonstration of acute calcification following transient ligation of the rodent MCA invites further studies.

Platelet-activating factor contributes to postischemic vasospasm.

BACKGROUND: The purpose of the present study was to determine if platelet-activating factor is an important mediator that produces vasospasm during reperfusion after ischemia in skeletal muscle. MATERIALS AND METHODS: A vascular isolated cremaster muscle in male Sprague-Dawley rats was coupled with local intraarterial drug infusion as a model to study microcirculation responses to ischemia/reperfusion injury. Arteriole diameters and capillary perfusion were measured using intravital microscopy. Group 1: platelet-activating factor dose response. Group 2: Effects of a cyclooxygenase inhibitor; indomethacin, and a thromboxane synthetase inhibitor, imidazole, on the response to platelet-activating factor. Group 3: Effects of nitric oxide synthesis inhibitor; N(omega)-nitro-L-arginine methyl ester, on the response to platelet-activating factor. Group 4: Effects of a platelet-activating factor receptor antagonist, CV-3988, indomethacin, and imidazole after 4 h of warm ischemia and reperfusion. RESULTS: Intraarterial infusion of platelet-activating factor produced a dose-related but mild vasoconstriction. Pretreatment with indomethacin or imidazole resulted in significant vasodilation actually emanating from platelet-activating factor infusion. Nitric oxide inhibition (with N(omega)-nitro-L-arginine methyl ester) enhanced the vasoconstriction produced by platelet-activating factor. Pretreatment with CV-3988, indomethacin, or imidazole significantly attenuated ischemia/reperfusion-induced vasospasm and capillary no-reflow in the cremaster muscles. CONCLUSIONS: Ischemia/reperfusion-induced vasoconstriction is at least in part mediated by platelet-activating factor and thromboxane A(2).

Carbon dioxide narcosis-induced apnea in a rat model of cardiac arrest and resuscitation.

In the clinical literature there are reports of patients failing to breathe and becoming comatose when supplied with 100% oxygen for respiratory distress. This effect has been attributed to a loss of respiratory drive. Recent studies have established that this explanation is incorrect, but have left the phenomenon unexplained. We propose that the apnea and coma reported is due to carbon dioxide narcosis. We have reproduced this effect in an animal model and have documented PCO2 values in excess of 250 mmHg during the apneic period. Our results suggest that this level of PCO2 suppresses both brainstem auditory evoked potentials and spontaneous respiration. The high PCO2 is due to inadequate gas exchange, and is easily remedied by provision of adequate ventilation.